Homogeneous assay of biopolymer binding by means of multiple measurements under varied conditions

ABSTRACT

A method for homogeneously assaying biopolymer bonding includes obtaining signals from a test sample before, during and/or after the application of stimulus to the test sample and correlating the signals. The signals, whose magnitude correlate with binding affinity, can be, for example, electrical conductance and/or fluorescent intensity. The stimulus can be, for example, electric voltage and/or laser radiation. Preferably, different types of signals are measured and compared so as to enhance the reliability of the assay.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/490,273, filed Jan. 24, 2000 now U.S. Pat. No. 6,265,170.

BACKGROUND OF THE INVENTION

1. Field of Invention

The invention relates to methods of assaying biopolymer binding, andmore particularly to methods of assaying binding of probes and targetscontaining nucleobases and/or amino acids.

2. Description of Related Art

It has been understood for a number of years that biological moleculescan be isolated and characterized through the application of an electricfield to a sample.

Electrophoresis is perhaps the most well-known example of an isolationand characterization technique based on the influence of electric fieldson biological molecules. In gel electrophoresis, a uniform matrix or gelis formed of, for example, polyacrylamide, to which an electric field isapplied. Mixtures applied to one end of the gel will migrate through thegel according to their size and interaction with the electric field.Mobility is dependent upon the unique characteristics of the substancesuch as conformation, size and charge. Mobilities can be influenced byaltering pore sizes of the gel, such as by formation of a concentrationor pH gradient, or by altering the composition of the buffer (pH, SDS,DOC, glycine, salt). One- and two-dimensional gel electrophoresis arefairly routine procedures in most research laboratories. Targetsubstances can be purified by passage through and/or physical extractionfrom the gel.

A more recently developed process in which an electric field is appliedto a biological sample is disclosed in U.S. Pat. No. 5,824,477 toStanley. The Stanley patent discloses a process for detecting thepresence or absence of a predetermined nucleic acid sequence in asample. The process comprises: (a) denaturing a sample double-strandednucleic acid by means of a voltage applied to the sample in a solutionby means of an electrode; (b) hybridizing the denatured nucleic acidwith an oligonucleotide probe for the sequence; and (c) determiningwhether the hybridization has occurred. The Stanley patent discloses theapplication of an electric field to the sample to be assayed for thelimited purpose of denaturing the target sequence.

A more well-known type of hybridization assay is based on the use offluorescent marking agents. In their most basic form, fluorescentintensity-based assays have typically comprised contacting a target witha fluorophore-containing probe, removing any unbound probe from boundprobe, and detecting fluorescence in the washed sample. Homogeneousassays improve upon such basic assays, in that the former do not requirea washing step or the provision of a non-liquid phase support.

Some assays have employed intercalating fluorophores to detect nucleicacid hybridization, based on the ability of such fluorophores to bindbetween strands of nucleic acid in a hybridization complex.

For example, U.S. Pat. No. 5,824,557 to Burke et al. discloses a methodand kit for detecting and quantitating nucleic acid molecules. Apreferred embodiment relies on the intercalation of a dye into adouble-stranded nucleic acid helix or single-stranded nucleic acid. Thedye fluoresces after intercalation and the intensity is a directmeasurement of the amount of nucleic acid present in the sample. Whilethe method of Burke et al. is purported to be useful for measuring theamount of nucleic acid in a sample, the non-specific binding betweenintercalator and nucleic acid upon which the method is based renders themethod impractical for detecting specific binding, particularly underconditions where non-target nucleic acid duplexes are present.

U.S. Pat. No. 5,814,447 to Ishiguro et al. discloses an assay which ispurported to improve upon assays that rely on non-specific interactionbetween intercalating agents and nucleic acid duplexes, such as Burke etal. and an earlier assay described by Ishiguro et al. in Japanese PatentPublic Disclosure No. 237000/1993. The earlier development comprisedadding an intercalating fluorochrome having a tendency to exhibitincreased intensity of fluorescence when intercalated to a samplesolution before a specific region of a target nucleic acid was amplifiedby PCR, and measuring the intensity of fluorescence from the reactionsolution at given time intervals to detect and quantitate the targetnucleic acid before amplification. The '447 patent attempted to improveupon the earlier development by providing an assay having improvedspecificity, characterized in that the probe is a single-strandedoligonucleotide labeled with an intercalating fluorochrome which is tobe intercalated into a complementary binding portion between a targetnucleic acid and a single-stranded oligonucleotide probe.

In the ongoing search for more sensitive, accurate and rapid assaytechniques, one research group developed an assay comprising analyzingthe effects of an electric field on the fluorescent intensity of nucleicacid hybridization duplexes. See U.S. patent application Ser. No.08/807,901, filed Feb. 27, 1997 and U.S. Pat. No. 6,060,242. Theresearchers indicated that the fluorescent intensity of a one base-pairmismatched duplex differed from that of a perfectly matched duplex.Thus, the applications purport to disclose a method for detecting anucleotide sequence, wherein an electric field is applied to a liquidmedium prior to or concurrently with a detecting step, and a change inan intensity of a fluorescent emission as a function of the electricfield is detected as an indication of whether the probe is hybridized toa completely complementary nucleotide sequence or an incompletelycomplementary nucleotide sequence.

Despite the foregoing developments, a need has continued to exist in theart for a simple, highly sensitive, effective and rapid method foranalyzing interaction between nucleic acids and/or nucleic acid analogs.

All references cited herein are incorporated herein by reference intheir entireties.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method for assaying sequence-specifichybridization, which comprises:

-   -   providing a target comprising at least one target biopolymer        sequence;    -   providing a probe comprising at least one probe biopolymer        sequence;    -   adding the probe and the target to a binding medium to provide a        test sample;    -   applying a first stimulus to the test sample to provide a first        stimulated test sample;    -   detecting a first signal from the first stimulated test sample,        wherein the first signal is correlated with a binding affinity        between the probe and the target;    -   applying a second stimulus to the first stimulated test sample        to provide a second stimulated test sample;    -   detecting a second signal from the second stimulated test        sample, wherein the second signal is correlated with the binding        affinity between the probe and the target; and    -   comparing the first signal and the second signal to accomplish        the assaying;    -   wherein at least one label is provided in the test sample, and        the first stimulus, the second stimulus, the first signal and        the second signal are electromagnetic radiation, provided that        when the first stimulus and the second stimulus are photonic        radiation, an intermediate electronic stimulus is applied to the        test sample after the first stimulus and before the second        stimulus, and when the first stimulus and the second stimulus        are electronic radiation, the first signal and the second signal        are electric current.

Also provided is a method for assaying sequence-specific hybridization,the method comprising:

-   -   providing a target;    -   providing a probe, wherein at least one of the probe and the        target comprises at least one biopolymer sequence;    -   adding the probe and the target to a binding medium to provide a        test sample;    -   applying a first stimulus to the test sample to provide a first        stimulated test sample;    -   detecting a first signal from the first stimulated test sample,        wherein the first signal is correlated with a binding affinity        between the probe and the target;    -   applying a second stimulus to the first stimulated test sample        to provide a second stimulated test sample;    -   detecting a second signal from the second stimulated test        sample, wherein the second signal is correlated with the binding        affinity between the probe and the target; and    -   comparing the first signal and the second signal to accomplish        the assaying;

wherein at least one label is provided in the test sample, and the firststimulus, the second stimulus, the first signal and the second signalare electromagnetic radiation, provided that when the first stimulus andthe second stimulus are photonic radiation, an intermediate electronicstimulus is applied to the test sample after the first stimulus andbefore the second stimulus, and when the first stimulus and the secondstimulus are electronic radiation, the first signal and the secondsignal are electric current.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The invention will be described in conjunction with the followingdrawings in which like reference numerals designate like elements andwherein:

FIGS. 1A and 1B are graphs of current as a function of time andcomplementarity;

FIGS. 1C and 1D are graphs of current as a function of temperature andcomplementarity;

FIGS. 2A, 2B, 2C, 3A and 3B are graphs of current as a function oftemperature, complementarity and additional factors;

FIG. 4 is a graph of current as a function of time and complementarity;and

FIGS. 5A, 5B, 5C and 6 are fluorescent intensity spectra.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a rapid, sensitive, environmentally friendly, andsafe method for assaying binding between a target and a probe, whereinthe target comprises a nucleic acid sequence or a nucleic acid analogsequence and the probe comprises a nucleic acid sequence or a nucleicacid analog sequence. The assay of the invention is also suitable forassaying binding between a target and a probe, wherein the target and/orthe probe comprises an amino acid sequence. Thus, the invention issuitable for assaying binding of biopolymers, which as used herein,means a sequence containing at least two amino acids, amino acidanalogs, nucleic acids, nucleic acid analogs and/or combinationsthereof.

Unlike certain prior art assays, the invention not only detects thepresence of specific binding, but also provides qualitative andquantitative information regarding the nature of binding between a probeand target. Thus, in embodiments comprising nucleobase to nucleobasebinding assays, the invention enables the practitioner to distinguishamong a perfect match, a one base pair mismatch, a two base pairmismatch, a three base pair mismatch, a one base pair deletion, a twobase pair deletion and a three base pair deletion.

Embodiments of the invention comprise calibrating the measured signal(e.g., electric current and/or fluorescent intensity) for a firstprobe-target mixture against the same type of signal exhibited by otherprobes combined with the same target, wherein each of the other probesdiffers from the first probe by at least one base.

In certain embodiments, a low voltage is applied to the sample prior toor concurrent with measuring said signal. Generally, the voltage isselected such that it is high enough to destabilize imperfectly matchedhybridization partners but not so high as to destabilize perfectlymatched hybridization partners. In certain preferred embodiments, thevoltage is about 1V to about 20V.

A calibration curve can be generated, wherein the magnitude of themeasured signal (e.g., electric current and/or fluorescent intensity) isa function of the binding affinity between the target and probe. As thebinding affinity between the target and a plurality of different probesvaries with the number of mismatched bases in nucleobase-nucleobaseassays, the nature of the mismatch (A-G vs. A-C vs. T-G vs. T-C, etc.),the location of the mismatch(es) within the hybridization complex, etc.,the assay of the invention can be used to sequence the target.

The signal measured can be, e.g., electrical conductance. In suchembodiments, the binding affinity between the probe and target isdirectly correlated with the magnitude of the signal. That is, theelectrical conductance increases along with the extent of matchingbetween the probe and target, preferably over a range inclusive of 0-2mismatches and/or deletions, more preferably over a range inclusive of0-3 mismatches and/or deletions.

In other embodiments, the signal measured can be the fluorescentintensity of a fluorophore included in the test sample. In suchembodiments, the binding affinity between the probe and target can bedirectly or inversely correlated with the intensity, depending onwhether the fluorophore signals hybridization through signal quenchingor signal amplification. Thus, the fluorescent intensity generated byintercalating agents is directly correlated with probe-target bindingaffinity, whereas the intensity of embodiments employingnon-intercalating fluorophores covalently bound to the probe isinversely correlated with probe-target binding affinity. The fluorescentintensity increases (or decreases for non-intercalators) along with theextent of matching between the probe and target, preferably over a rangeinclusive of 0-2 mismatches and/or deletions, more preferably over arange inclusive of 0-3 mismatches and/or deletions.

Although the inventors have previously disclosed the advantages offluorescent intensity assays for analyzing hybridization ofnucleobase-containing sequences (see U.S. patent application Ser. No.09/468,679, filed Dec. 21, 1999) and the advantages of fluorescentintensity assays for analyzing peptide:nucleic acid binding (see U.S.patent application Ser. No. 09/224,505, filed Dec. 31, 1998) andpeptide:peptide binding (see U.S. patent application Ser. No.09/344,525, filed Jun. 25, 1999), the application of an electric fieldto the sample appears to increase the resolution of the assay, as shownin Example 6 below.

Moreover, in particularly preferred embodiments of the invention, theassay comprises measuring at least two signals of the sample. The firstsignal is preferably fluorescent intensity and the second signal ispreferably selected from several electrical conductance measurements (orvice versa).

In the preferred multiple measurement embodiments, the first signal canbe the same as or different from the second signal. When the first andsecond signals measured are the same, the second signal can becalibrated against the first signal and/or against the same referencesignal(s) used to calibrate the first signal. In addition, at least onecondition-altering stimulus is preferably applied to the test sampleafter the first signal is measured and before the second signal ismeasured. The stimulus is preferably sufficient to measurably changebinding, as indicated by at least one signal. In nucleobase-nucleobasebinding assays of the invention, the stimulus is preferably sufficientto significantly affect imperfectly complementary hybridization betweenthe probe and the target and insufficient to significantly affectperfectly complementary hybridization between the probe and the target.

In certain embodiments of the invention, at least one stimulus isapplied once or a plurality of times. The stimulus can be continuouslyapplied or non-continuously applied. The stimulus can be applied before,during and/or after the detection of signal detection.

Suitable stimuli can be, e.g., photonic radiation (such as laser light)and/or electronic. The signals detected can be, e.g., photonic and/orelectronic as well.

For example, in a particularly preferred embodiment of the invention,the first signal measured is pre-electrification fluorescent intensity(i.e., intensity measured before a condition-altering voltage is appliedto the test sample) and the second signal measured ispost-electrification fluorescent intensity (i.e., intensity measuredduring or after the condition-altering voltage has been applied to thetest sample)

The additional measurements in the foregoing embodiments increase thereliability of the assay and enable immediately retesting suspectresults. Inconsistent results achieved by the at least two measurementswill typically warrant retesting.

The invention enables quantifying the binding affinity between probe andtarget. Such information can be valuable for a variety of uses,including designing antisense drugs with optimized bindingcharacteristics.

Unlike prior art methods, the assay of the invention is preferablyhomogeneous. The assay can be conducted without separating theprobe-target complex from the free probe and target prior to detectingthe magnitude of the measured signal. The assay does not require a gelseparation step, thereby allowing a great increase in testingthroughput. Quantitative analyses are simple and accurate. Consequentlythe binding assay saves a lot of time and expense, and can be easilyautomated. Furthermore, it enables binding variables such as buffer, pH,ionic concentration, temperature, incubation time, relativeconcentrations of probe and target sequences, intercalatorconcentration, length of target sequences, length of probe sequences,and possible cofactor requirements to be rapidly determined.

The assay can be conducted in e.g., a solution within a well, on animpermeable surface or on a biochip.

Moreover, the inventive assay is preferably conducted without providinga signal quenching agent on the target or on the probe.

Preferred embodiments of the invention specifically detect triplexand/or quadruplex hybridization between the probe and thedouble-stranded target, thus obviating the need to denature the target.Triplex and quadruplex formation and/or stabilization is enhanced by thepresence of an intercalating agent in the sample being tested. See,e.g., U.S. patent application Ser. No. 09/885,731, filed Jun. 20, 2001,and the U.S. Patent Application Ser. No. 09/909,496, entitled “PARALLELOR ANTIPARALLEL, HOMOLOGOUS OR COMPLEMENTARY BINDING OF NUCLEIC ACIDS ORANALOGUES THEREOF TO FORM DUPLEX, TRIPLEX OR QUADRUPLEX COMPLEXES”,filed Jul. 20, 2001.

Complexes of the invention preferably do not rely on Hoogsteen bondingor G-G quartets for maintenance of the complex structure, althoughHoogsteen bonding and/or G-G quartets may be present. That is, complexesof the invention are preferably substantially free of Hoogsteen bonding,and substantially free of G-G quartets.

Suitable nucleobase-containing probes for use in the inventive assayinclude, e.g., ssDNA, RNA, PNA and other nucleic acid analogs havinguncharged or partially-charged backbones. Although antiparallel probesare preferred in certain embodiments, probes can also be parallel. Probesequences having any length from 8 to 20 bases are preferred since thisis the range within which the smallest unique DNA sequences ofprokaryotes and eukaryotes are found. Probes of 12 to 18 bases areparticularly preferred since this is the length of the smallest uniquesequences in the human genome. In embodiments, probes of 6 to 30 basesare most preferred. However, a plurality of shorter probes can be usedto detect a nucleotide sequence having a plurality of non-unique targetsequences therein, which combine to uniquely identify the nucleotidesequence. The length of the probe can be selected to match the length ofthe target.

Suitable amino acid-containing probes can comprise a single amino acid,single amino acid analog, a peptide-like analog, peptidoid,peptidomimetic, peptide, dipeptide, tripeptide, polypeptide, protein ora multi-protein complex.

The invention does not require the use of radioactive probes, which arehazardous, tedious and time-consuming to use, and need to be constantlyregenerated. Probes of the invention are preferably safe to use andstable for years. Accordingly, probes can be made or ordered in largequantities and stored.

In embodiments of the invention wherein the target comprises aminoacids, the target preferably comprises a peptide sequence or apeptide-like analog sequence, such as, e.g., a dipeptide, tripeptide,polypeptide, protein or a multi-protein complex. More preferably, thetarget is a protein having at least one receptor site for the probe.

In embodiments of the invention wherein the target comprisesnucleobases, the targets are preferably 8 to 3.3×109 base pairs long,and can be single or double-stranded sequences of nucleic acids and/oranalogs thereof.

It is preferred that the probe and target be unlabeled, but inalternative embodiments, there is an intercalating agent covalentlybound to the probe. In such embodiments, the intercalating agent ispreferably bound to the probe at either end.

In other embodiments, the intercalating agent is not covalently bound tothe probe, although it can insert itself between the probe and targetduring the assay, in a sense bonding to the probe in a non-covalentfashion.

Preferred intercalating agents for use in the invention include, e.g.,YOYO-1, TOTO-1, ethidium bromide, ethidium homodimer-1, ethidiumhomodimer-2 and acridine. In general, the intercalating agent is amoiety that is able to intercalate between strands of a duplex, triplexand/or a quadruplex nucleic acid complex. In preferred embodiments, theintercalating agent (or a component thereof) is essentiallynon-fluorescent in the absence of nucleic acids and fluoresces whenintercalated and excited by radiation of an appropriate wavelength,exhibiting a 100-fold to 10,000-fold enhancement of fluorescence whenintercalated within a duplex or triplex nucleic acid complex.

In alternative embodiments, the intercalating agent may exhibit a shiftin fluorescent wavelength upon intercalation and excitation by radiationof an appropriate wavelength. The exact fluorescent wavelength maydepend on the structure of the nucleic acid that is intercalated, forexample, DNA vs. RNA, duplex vs. triplex, etc.

The excitation wavelength is selected (by routine experimentation and/orconventional knowledge) to correspond to this excitation maximum for thefluorophore being used, and is preferably 200 to 1000 nm. Intercalatingagents are preferably selected to have an emission wavelength of 200 to1000 nm. In preferred embodiments, an argon ion laser is used toirradiate the fluorophore with light having a wavelength in a range of400 to 540 nm, and fluorescent emission is detected in a range of 500 to750 nm.

The assay of the invention can be performed over a wide variety oftemperatures, such as, e.g., from 5 to 85° C. Certain prior art assaysrequire elevated temperatures, adding cost and delay to the assay. Onthe other hand, the invention can be conducted at room temperature orbelow (e.g., at a temperature below 25° C.).

The inventive assay is extremely sensitive, thereby obviating the needto conduct PCR amplification of the target. For example, in at least thefluorescent intensity embodiments, it is possible to assay a test samplehaving a volume of about 20 microliters, which contains about 10femtomoles of target and about 10 femtomoles of probe. Embodiments ofthe invention are sensitive enough to assay targets at a concentrationof 5×10⁻⁹ M, preferably at a concentration of not more than 5×10⁻¹⁰ M.Embodiments of the invention are sensitive enough to employ probes at aconcentration of 5×10⁻⁹ M, preferably at a concentration of not morethan 5×10⁻¹⁰ M.

Conductivity measurements can distinguish samples having as little asabout 1 pmole of probe and 1 pmole of target in 40 microliters.Decreasing the sample volume would permit the use of even smalleramounts of probe and target.

It should go without saying that the foregoing values are not intendedto suggest that the method cannot detect higher concentrations.

A wide range of intercalator concentrations are tolerated at eachconcentration of probe and target tested. For example, when 5×10⁻¹⁰ Mprobe and 5×10⁻¹⁰ M target are hybridized, the optimal concentration ofthe intercalator YOYO-1 ranges from 25 nM to 2.5 nM. At a 5×10⁻⁸ Mconcentration of both probe and target, the preferred YOYO-1concentration range is 1000 nM to 100 nM.

The assay is sufficiently sensitive to distinguish a one base-pairmismatched probe-target complex from a two base-pair mismatchedprobe-target complex, and preferably a two base-pair mismatchedprobe-target complex from a three base-pair mismatched probe-targetcomplex. Of course, the assay is sufficiently sensitive to distinguish aperfectly matched probe-target complex from any of the above mismatchedcomplexes.

The binding medium can be any conventional medium known to be suitablefor preserving nucleotides and/or proteins. See, e.g., Sambrook et al.,“Molecular Cloning: A Lab Manual,” Vol. 2 (1989). For example, theliquid medium can comprise nucleotides, water, buffers and standard saltconcentrations.

Hybridization between complementary bases occurs under a wide variety ofconditions having variations in temperature, salt concentration,electrostatic strength, and buffer composition. Examples of theseconditions and methods for applying them are known in the art.

It is preferred that hybridization complexes be formed at a temperatureof about 15° C. to about 25° C. for about 1 minute to about 5 minutes.Longer reaction times are not required, but incubation for several hourswill not adversely affect the hybridization complexes.

It is possible (although unnecessary, particularly for embodimentscontaining an intercalating agent) to facilitate hybridization insolution by using certain reagents. Preferred examples of these reagentsinclude single stranded binding proteins such as Rec A protein, T4 gene32 protein, E. coli single stranded binding protein, major or minornucleic acid groove binding proteins, divalent ions, polyvalent ions,viologen and intercalating substances such as ethidium bromide,actinomycin D, psoralen, and angelicin. Such facilitating reagents mayprove useful in extreme operating conditions, for example, underabnormal pH levels or extremely high temperatures.

The inventive assay can be used to, e.g., identify accessible regions infolded nucleotide sequences, to determine the number of mismatched basepairs in a hybridization complex, and to map genomes.

In embodiments wherein fluorescent intensity is detected using anintercalating agent, intensity increases with increasing bindingaffinity between the probe and target. In embodiments whereinfluorescent intensity is detected using a non-intercalating fluorophore,intensity decreases as binding affinity increases between the probe andtarget. Regardless of whether the fluorophore intercalates or not, theinstant method does not require the measurement of the polarization offluorescence, unlike fluorescent anisotropy methods.

The invention will be illustrated in more detail with reference to thefollowing Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1

Sense and antisense 50-mer ssDNA target sequences, derived from exon 10of the human cystic fibrosis gene (Nature 380, 207 (1996)) weresynthesized on a DNA synthesizer (Expedite 8909, PerSeptive Biosystems)and purified by HPLC. Equimolar amounts of complementaryoligonucleotides were denatured at 95° C. for 10 min and allowed toanneal gradually as the temperature cooled to 21° C. over 1.5 hours.Double stranded DNA (dsDNA) oligonucleotides were dissolved in ddH₂O ata concentration of 1 pmole/μl.

Sequence for the sense strand of the wild-type target DNA

(SEQ ID NO: 1): 5′-TGG CAC CAT TAA AGA AAA TAT CAT CTT TGG TGT TTC CTATGA TGA ATA TA-3′.

Sequence for the antisense strand of the wild-type target

DNA (SEQ ID NO: 1): 5′-TAT ATT CAT CAT AGG AAA CAC CAA AGA TGA TAT TTTCTT TAA TGG TGC CA-3′.

The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO:1) is65.2° C.

SEQ ID NO:2 was a 50-mer mutant dsDNA target sequence identical towild-type target DNA (SEQ ID NO:1) except for a one base pair mutation(underlined) at amino acid position 507 at which the wild-type sequenceCAT was changed to CGT.

Sequence for the sense strand of

SEQ ID NO: 2: 5′-TGG CAC CAT TAA AGA AAA TAT CGT CTT TGG TGT TTC CTA TGATGA ATA TA-3′.

Sequence for the antisense strand of

SEQ ID NO: 2: 5′-TAT ATT CAT CAT AGG AAA CAC CAA AGA CGA TAT TTT CTT TAATGG TGC CA-3′.

The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO:2) is66.0° C.

SEQ ID NO:3 was a 50-mer mutant dsDNA target sequence identical towild-type target DNA (SEQ ID NO:1) except for a consecutive two basepair mutation (underlined) at amino acid positions 506 and 507 at whichthe wild-type sequence CAT was changed to ACT.

Sequence for the sense strand of

SEQ ID NO: 3: 5′-TGG CAC CAT TAA AGA AAA TAT ACT CTT TGG TGT TTC CTA TGATGA ATA TA-3′.

Sequence for the antisense strand of

SEQ ID NO: 3: 5′-TAT ATT CAT CAT AGG AAA CAC CAA AGA GTA TAT TTT CTT TAATGG TGC CA-3′.

The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO:3) is65.2° C.

The PNA probes used in the Examples were synthesized, HPLC purified andconfirmed by mass spectroscopy by Commonwealth Biotechnologies, Inc.(Richmond, Va., USA). PNA probes were first dissolved in 0.1% TFA(trifluoroacetic acid) to a concentration of 10 mg/ml, and then dilutedto 1 mg/ml by the addition of ddH₂O. Final PNA stock solutions wereprepared in ddH₂O at a concentration of 1 pmole/μl.

Probe No. 1 was a 15-mer antiparallel PNA probe designed to becompletely complementary to a 15 nucleotide segment of the sense strandof the 50-mer wild-type target DNA (SEQ ID NO:1), overlapping amino acidpositions 505 to 510 (Nature 380, 207 (1996)). The probe had thefollowing structure (SEQ ID NO:8):5′-H-CAC CAA AGA TGA TAT-Lys-CONH₂-3′

The hybridization reaction mixture (80 μl) contained the following: 2pmoles of target dsDNA, 2 pmoles of PNA probe, 0.5×TBE and 250 nM of theDNA intercalator YOYO-1 (Molecular Probes, Eugene, Oreg., USA). Sampleswere placed into a 3 mm quartz cuvette and were subjected to 1 or 5volts DC (V) electrification for 15 seconds. The amperometric assayconsisted of the monitoring of current while the voltage was beingapplied to the solution. A temperature probe was placed in each solutionto measure temperature at the time of amperometric assessment. At 1volt, a current peak was observed during the first 2 seconds ofelectrification. The current declined sharply over the following 13seconds. Experiments applying 5 volts gave rise to currents thatremained relatively stable over the entire electrification period (15seconds).

A series of experiments were carried out where the conductance valueswere observed when no DNA or PNA was present (control), or whenwild-type SEQ ID NO:1, mutant SEQ ID NO:2 or mutant SEQ ID NO:3 werereacted with antiparallel PNA Probe No. 1. FIGS. 1A and 1B plot the dataobtained for conductance in the individual experiments. FIG. 1A displaysthe results of the application of 1V electrification and FIG. 1B theapplication of 5V. Double stranded DNA:PNA hybrid triplexes consistingof perfectly complementary sequences (SEQ ID NO:1+Probe No. 1) allowedmaximum intercalation of YOYO-1, yielding the highest conductance values(depicted on the figures as negative current values) throughout theentire 15 seconds of 1V application. The normalized peak conductance forthe triplex hybridization of the antiparallel PNA probe with a 1 bpmismatched dsDNA (SEQ ID NO:2+Probe No. 1) and with the 2 bp mismatcheddsDNA (SEQ ID NO:3+Probe No. 1) were respectively 79% and 96% lower thanthat observed with the perfectly matched dsDNA:PNA triplex hybrid (SEQID NO: 1+Probe No. 1) during the first second of voltage application(FIG. 1A). Similar percent decreases in conductance between perfectlycomplementary triplexes and triplexes containing base pair mismatcheswere obtained when the conductance values over the entire 15 seconds ofvoltage application were averaged. In FIG. 1A the 1 bp and 2 bpmismatched dsDNA:PNA hybrids resulted in average conductance values thatwere 65% and 91% lower, respectively, than those for the perfectlymatched dsDNA:PNA hybrid. All experiments expressed in FIG. 1A werecarried out at room temperature (23° C.). As the degree of mismatchbetween the probe and the double stranded target increased, the level ofintercalation by YOYO-1 diminished and the level of conductancedecreased. These relationships were also observed when the experimentsreferred to above were repeated and a higher voltage (5V) was applied.During the 5V application the normalized average conductance values forthe 1 bp mismatched dsDNA:PNA triplex (SEQ ID NO:2+Probe No. 1) and the2 bp mismatched dsDNA:PNA triplex (SEQ ID NO:3+Probe No. 1) wererespectively 52% and 67% lower than that observed for the perfectlymatched dsDNA:PNA triplex (SEQ ID NO:3+Probe No. 1) (FIG. 1B).Experiments expressed in FIG. 1B were performed at room temperature (23°C.).

When the experiments were repeated with the temperature increased to 50°C. and 65° C., similar amperometric values were observed. At 50° C., theapplication of 1V for 15 seconds to the perfectly matched dsDNA:PNAtriplex (SEQ ID NO:1+Probe No. 1) produced an average current of −0.25μAmp as compared to values of −0.15 μAmp (a 40% reduction) and −0.06μAmp (a 76% reduction) for the 1 bp mismatched dsDNA:PNA triplex (SEQ IDNO:2+Probe No. 1) and the 2 bp mismatched dsDNA:PNA triplex (SEQ IDNO:3+Probe No. 1), respectively (FIG. 1C). At 65° C., similarobservations were noted when 1 V of electricity was applied for 15seconds. Perfectly matched nucleic acid hybrids produced an averagecurrent of −0.37 μAmp compared with −0.16 μAmp (a 57% reduction) and−0.01 μAmp (a 97% reduction) for 1 bp and 2 bp mismatched hybrids,respectively (FIG. 1C). The application of 5 volts at high temperaturesproduced analogous results. While experiments performed at 50° C.generated average currents of −0.27 mAmp, −0.13 mAmp (a 52% reduction),and −0.08 mAmp (a 70% reduction), for perfectly matched hybrids, 1 bpmismatched hybrids, and 2 bp mismatched hybrids, respectively,experiments performed at 65° C. resulted in average current values of−0.31 mAmp, −0.14 mAmp (a 55% reduction), and −0.10 mAmp (a 68%reduction) for the same three respective groups (FIG. 1D). For all ofthe foregoing experiments, dsDNA was not denatured prior to triplexhybridization with the antiparallel PNA Probe No. 1.

Similar experiments were done at varying temperatures after thehybridization mixes had been heated to 65° C. and immediately allowed tocool. After cooling to room temperature (23° C.), applying 1V for 15seconds to the perfectly matched sample (SEQ ID NO:1+Probe No. 1)produced an average current of −0.18 μAmp. By comparison, values of−0.06 μAmp (a 67% reduction) and −0.05 μAmp (a 72% reduction) for the 1bp mismatched dsDNA:PNA triplex hybrid (SEQ ID NO:2+Probe No. 1) and the2 bp mismatched dsDNA:PNA triplex hybrid (SEQ ID NO:3+Probe No. 1), wererespectively observed (data not shown). When the samples were cooledfrom 65° C. to 50° C., similar observations were noted when 1 V wassubsequently applied for 15 seconds. The perfectly matched sample (SEQID NO: 1+Probe No. 1) produced an average current of −0.23 μAmp comparedwith −0.11 μAmp (a 52% reduction) and −0.01 μAmp (a 96% reduction)observed for the 1 bp and 2 bp mismatched samples, respectively (datanot shown). When 5V was applied after cooling to 23° C. or 50° C., theaverage current generated in the perfectly matched triplex hybrid (SEQID NO:1+Probe No. 1), the 1 bp mismatched triplex hybrid (SEQ IDNO:2+Probe No. 1), and the 2 bp mismatched triplex hybrid (SEQ IDNO:3+Probe No. 1) were: −0.15 mAmp, −0.09 mAmp (a 40% reduction), and−0.07 mAmp (a 53% reduction), respectively at 23° C., and −0.23 mAmp,−0.09 mAmp (a 61% reduction), and −0.09 mAmp (a 61% reduction),respectively at 50° C. (data not shown).

Pretreatment of hybridization mixes at 65° C. (the T_(m) of the 50-merdsDNA sequences) followed by cooling did not significantly affect thedifference in conductance observed between perfectly complementarydsDNA:PNA triplexes and those containing 1 or 2 bp mismatches whenmeasured directly at 23° C. or 50° C. (without preheating at 65° C.)when an antiparallel PNA probe was used. Clearly, the antiparallel PNAprobe in the presence of the DNA intercalator YOYO-1 was able to formtriplex structures with the dsDNA targets. Application of low levels ofelectricity (such as 1V or 5V) allowed the perfectly matched dsDNA:PNAtriplex sequences to be distinguished from those containing 1 bp or 2 bpmutations, without prior denaturation of sequences.

Example 2

FIG. 2 demonstrates that the amperometric assay of the invention canalso discriminate between perfectly matched dsDNA:PNA triplex hybridsand those containing 1 bp or 2 bp mismatches when the PNA probe used isin a parallel orientation with respect to the target DNA sequence. ProbeNo. 2 was a 15-mer PNA probe identical in sequence to Probe No. 1, butwas synthesized to match the parallel orientation of the target DNA,instead of the conventional anti-parallel orientation. Probe No. 2 hadthe following structure (SEQ ID NO:9):5′-H-TAT AGT AGA AAC CAC-Lys-CONH₂-3′

Experiments with assay conditions identical to those described inExample 1 were carried out with the sole difference that Probe No. 2 wasused in place of Probe No. 1. When 1 volt was applied, the averagecurrent for a 1 bp mismatched dsDNA:PNA triplex (SEQ ID NO: 2+Probe No.2), and a consecutive 2 bp mismatched dsDNA:PNA triplex (SEQ ID NO:3+Probe No. 2), were respectively 25% and 32% lower at 23° C.,respectively 30% and 23% lower at 50° C., and respectively 28% and 53%lower at 65° C. than that observed with the perfectly matched dsDNA:PNAtriplex (SEQ ID NO:1+Probe No. 2) at matching temperatures (FIG. 2A).

Similar results were obtained when 5 V (instead of 1V) was applied for15 seconds. Perfectly matched dsDNA:PNA hybrids at 23° C., 50° C. and65° C. generated average currents of −0.15 mAmp, −0.24 mAmp and −0.17mAmp, respectively (FIG. 2B). Incompletely complementary triplexes witha 1 bp mismatch and a 2 bp mismatch produced average currents that were27% less (−0.11 mAmp) and 53% less (−0.07 mAmp), respectively at 23° C.,21% less (−0.19 mAmp) and 46% less (−0.13 mAmp), respectively at 50° C.,and unchanged (−0.17mAmp) and 18% less (−0.14 mAmp), respectively at 65°C., than that achieved by the perfectly matched hybrid samples (FIG.2B).

The results illustrated in FIGS. 2A and 2B indicated that when theparallel PNA Probe No. 2 was used, the differences in conductivityobtained between perfectly matched dsDNA:PNA triplexes and thosecontaining 1 bp or 2 bp mismatches were less dramatic than that achievedwith the antiparallel PNA Probe No. 1 (FIG. 1).

However, experiments involving parallel Probe No. 2 and the applicationof 5 V after the samples have been heated to 65° C. and immediatelyallowed to cool disclosed amperometric measurements which demonstratedenhanced signaling differences between perfectly matched dsDNA:PNAtriplexes and the 1 bp or 2 bp mismatched dsDNA:PNA triplexes (FIG. 2C).The perfectly matched hybrids (SEQ ID NO:1+Probe No. 2), the 1 bpmismatched hybrids (SEQ ID NO:2+Probe No. 2) and the 2 bp mismatchedhybrids (SEQ ID NO:3+Probe No. 2) yielded average conductance values of−0.19 mAmps, −0.08 mAmps and −0.06 mAmps, respectively at 23° C., −0.17mAmps, −0.09 mAmps and −0.07 mAmps, respectively at 50° C., and −0.23mAmps, −0.13 mAmps and −0.08 mAmps, respectively at 65° C. Thistranslated to reductions in conductivity of 58% and 35 68% at 23° C.,47% and 59% at 50° C., and 43% and 65% at 65° C. for the 1 bp and 2 bpmismatched samples, respectively, when compared to the values achievedby the perfectly complementary samples (FIG. 2C).

Therefore, both antiparallel and parallel PNA probes in the amperometricassay are capable of discriminating between perfectly complementarydsDNA targets and incompletely complementary dsDNA targets containing 1bp or 2 bp mutations.

Example 3

Probe No. 3 was a 15-mer ssDNA probe identical in sequence andorientation to the 15-mer antiparallel PNA Probe No. 1 (SEQ ID NO:8).Probe No. 3 had the following structure:5′-CAC CAA AGA TGA TAT-3′

The specificity of the amperometric assay was further investigated byreacting ssDNA Probe No. 3 with the 50-mer wild-type and mutant dsDNAtarget sequences in the absence of prior denaturation. The assayconditions were identical to that described in Example 1.

Enhanced by the DNA intercalator YOYO-1, dsDNA:ssDNA triplexes wereformed between 30° C. and 65° C. Upon 1 volt treatment, the perfectlymatched DNA triplex, consisting of SEQ ID NO:1+Probe No. 3, yielded thehighest conductivity values (FIG. 3A). In contrast, incompletelycomplementary probe and target combinations generating a 1 bp mismatch(SEQ ID NO:2+Probe No. 3), and a consecutive 2 bp mismatch (SEQ IDNO:3+Probe No. 3), resulted in average conductance values that were 14%and 64% lower at 23° C., 30% and 70% lower at 50° C., and 25% and 72%lower at 65° C., respectively, than that observed with the perfectlycomplementary sequences at matching temperatures (FIG. 3A). Theapplication of a higher voltage (5V) to these samples resulted ingreater amperometric differences observed between perfectly matched andmismatched samples, than that obtained at 1 V, particularly at lowertemperatures. After a 5V treatment for 15 seconds, the average currentsfor the 1 bp mismatched DNA triplex and the 2 bp mismatched DNA triplexwere 54% and 78% lower, respectively at 23° C., 68% and 70% lower,respectively at 50° C., and 33% and 61% lower, respectively at 65° C.,than that observed with the perfectly matched DNA triplex at matchingtemperatures (FIG. 3B).

In similar electricity experiments, the hybridization mixes were heatedto 65° C. and were either maintained at this temperature or immediatelyallowed to cool to 50° C. or 23° C. prior to application of 1V or 5V. A1V treatment for 15 seconds to the perfectly matched DNA triplexsequences (SEQ ID NO:1+Probe No. 3) produced the highest conductancevalues at 23° C., 50° C. and 65° C. (FIG. 3A). The DNA triplexescontaining a 1 bp mismatch (SEQ ID NO:2+Probe No. 3) or a 2 bp mismatch(SEQ ID NO:3+Probe No. 3) were less conductive by 21% and 63%,respectively at 23° C., by 18% and 74%, respectively at 50° C., and by12% and 106%, respectively at 65° C. (FIG. 3A). Similarly, when 5V wereapplied for 15 seconds to pre-heated samples, the average conductancevalues for the 1 bp mismatched DNA triplexes and the 2 bp mismatched DNAtriplexes were reduced by 24% and 104%, respectively at 23° C., by 42%and 44%, respectively at 50° C., and by 38% and 102%, respectively at65° C., when compared to the average conductance values generated by theperfectly matched DNA triplexes (FIG. 3B).

The observation that the antiparallel PNA probe (FIG. 1) and ssDNA probe(FIG. 3) behaved in a similar fashion in the amperometric assay,suggested that the backbone of the nucleic acid entity used as the probewas not particularly important. The presence of YOYO-1 allowed the dsDNAtargets and the ssDNA probe to form a triple helix conformation capableof generating different electrical charges depending on the level ofsequence complementarity between the target and the probe in solution.As the degree of mismatch between the probe and the target increased,the level of conductance decreased, proving the reliability of theamperometric assay when a natural DNA probe was used in the absence ofprior denaturation.

Example 4

In the amperometric assays illustrated in Examples 1 to 3, the DNAintercalator YOYO-1 was added to the solution containing thehybridization mixes. Intercalation by YOYO-1 facilitated the formationof the dsDNA:PNA triplexes and dsDNA:ssDNA triplexes. The possibility ofutilizing an intercalator moiety covalently tethered to a ssDNA probe inthe amperometric assay was evaluated in Example 4.

Acridine is an alternative dsDNA intercalator, that also possesses theability to intercalate into triplex nucleic acid structures, therebystabilizing the triple helix formation.

See, e.g., Kukreti et al., “Extension of the range of DNA sequencesavailable for triple helix formation: stabilization of mismatchedtriplexes by acridine-containing oligonucleotides.” 25 Nucleic AcidsResearch 4264-4270 (1997). A ssDNA probe containing an acridine molecule(Glen Research, Sterling, Va., USA) covalently attached at the 3′ endwas synthesized on a DNA synthesizer (Expedite 8909, PerSeptiveBiosystems) and purified by HPLC.

Probe No. 4 was a 15-mer ssDNA probe identical in sequence andorientation to the 15-mer Probe No. 3 (and thus also identical insequence and orientation to the 15-mer antiparallel PNA Probe No. 1 (SEQID NO:8)) but with the addition of an acridine moiety at the 3′position. The probe had the following structure:5′-CAC CAA AGA TGA TAT-acridine-3′

The hybridization reaction mixture (80 μl) contained the following: 2pmoles of target dsDNA, 2 pmoles of ssDNA Probe No. 4 and 0.5×TBE.Samples were placed into a 3 mm quartz cuvette and were subjected to 5VDC electrification for 11 seconds at 23° C. The current and temperaturewere monitored as described in Example 1.

As shown in FIG. 4, the ssDNA Probe No. 4 was able to hybridize with the50-mer perfectly matched dsDNA target (SEQ ID NO:1) as a result of thestable intercalation of the covalently tethered acridine moiety,generating an average current of −0.53 mAmp. By comparison, the lessstable DNA triplexes containing a 1 bp mismatch (SEQ ID NO:2+Probe No.4) or a 2 bp mismatch (SEQ ID NO:3+Probe No. 4) produced averagecurrents that were 52% and 66% lower, respectively, than that achievedby the perfectly matched DNA triplex, when normalized against thecontrol (Probe No. 4 without target DNA) (FIG. 4).

Therefore, the acridine attached to a ssDNA probe was equally asefficient as untethered YOYO-1 in forming triple DNA helices thatgenerated different electrical currents depending on the level ofsequence complementarity between the target and the probe in theamperometric assay.

Example 5

Sense and antisense 15-mer ssDNA target sequences, derived from exon 10of the human cystic fibrosis gene, were synthesized, purified andannealed as described in Example 1. DsDNA oligonucleotides weredissolved in ddH₂0 at a concentration of 1 pmole/μl.

SEQ ID NO:4 was a 15-mer dsDNA target sequence derived from SEQ ID NO:1,designed to be completely complementary to Probe No. 1.

Sequence for the sense strand of the wild-type target DNA (SEQ ID NO:4):5′-ATA TCA TCT TTG GTG-3′.

Sequence for the antisense strand of the wild-type target DNA (SEQ IDNO:4):5′-CAC CAA AGA TGA TAT-3′.

The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO:4) is 40°C.

SEQ ID NO:5 was a 15-mer mutant dsDNA target sequence identical towild-type target DNA (SEQ ID NO:4) except for a one base pair mutation(underlined), at which the sequence TTT was changed to TAT.

Sequence for the sense strand of the mutant target DNA (SEQ ID NO:5):5′-ATA TCA TCT ATG GTG-3′.

Sequence for the antisense strand of the mutant target DNA (SEQ IDNO:5):5′-CAC CAT AGA TGA TAT-3′.

The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO:5) is40.0° C.

SEQ ID NO:6 was a 15-mer mutant dsDNA target sequence identical towild-type target DNA (SEQ ID NO:4) except for a consecutive two basepair mutation (underlined), at which the sequence ATC was changed toGGC.

Sequence for the sense strand of the mutant target DNA (SEQ ID NO:6):5′-ATA TCG GCT TTG GTG-3′.

Sequence for the antisense strand of the mutant target DNA (SEQ IDNO:6):5′-CAC CAA AGC CGA TAT-3′.

The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO:6) is44.0° C.

SEQ ID NO:7 was a 15-mer mutant dsDNA target sequence identical towild-type target DNA (SEQ ID NO:4) except for a separated three basepair mutation (underlined), wherein three 1 bp mutations were separatedby 3 base pairs each. The sequences ATC, TCT and TGG were changed toACC, TAT and TAG, respectively.

Sequence for the sense strand of the mutant target DNA (SEQ ID NO:7):5′-ATA CCA TAT TTA GTG-3′.

Sequence for the antisense strand of the mutant target DNA (SEQ IDNO:7):5′-CAC TAA ATA TGG TAT-3′.

The predicted melting temperature (T_(m)) of dsDNA (SEQ ID NO:7) is38.0° C.

The hybridization reaction mixture (80 μl) contained the following: 2pmoles of target dsDNA, 2 pmoles of parallel PNA Probe No. 2, 0.5×TBEand 250 nM of the DNA intercalator YOYO-1. The reaction mixtures wereincubated at 95° C. for 5-10 minutes to allow denaturation, and thenmaintained at 65° C. until assayed. Samples were placed into a quartzcuvette, irradiated with an argon ion laser beam having a wavelength of488 nm and monitored for fluorescent emission at 65° C. Concurrenttemperature measurements were achieved by a software-controlledtemperature probe placed directly into each sample. The maximumfluorescent intensity occurred at a wavelength of 536 nm, indicative ofintercalation of YOYO-1 in the PNA:DNA hybrids. As a second assay,following the initial laser irradiation of each sample, the same sampleswere subjected to 1V DC electrification for 4 seconds. During the finalsecond of electrification the samples were irradiated a second time withthe argon ion laser and monitored for fluorescent emission at 65° C.Fluorescent intensities were plotted as a function of wavelength foreach sample analyzed.

SsDNA:PNA hybrids consisting of perfectly complementary sequences (SEQID NO:4+Probe No. 2) allowed maximum intercalation of YOYO-1, yieldingthe highest fluorescent intensities (FIG. 5A). The fluorescentintensities for a 1 bp mismatched ssDNA:PNA hybrid (SEQ ID NO:5+ProbeNo. 2), a consecutive 2 bp mismatched ssDNA:PNA hybrid (SEQ IDNO:6+Probe No. 2), and a separated 3 bp mismatched ssDNA:PNA hybrid (SEQID NO:7+Probe No. 2) were all lower than that observed with theperfectly matched ssDNA:PNA hybrid at 65° C. (FIG. 5 and data notshown). As the degree of mismatch between the probe and the targetincreased, the level of intercalation by YOYO-1 diminished and hence thelevel of fluorescent intensity decreased. Only background levels offluorescence were observed when no DNA or PNA was present (YOYO-1 alone)(FIG. 5A).

When the perfectly matched ssDNA:PNA hybrids were subjected to 1V ofelectricity for 4 seconds at 65° C., the fluorescent intensity remainedrelatively constant, decreasing by only 2% (FIG. 5A). In contrast,application of 1V to the incompletely complementary duplexes containinga 1 bp mismatch (FIG. 5B), a 2 bp mismatch (FIG. 5C) and a 3 bp mismatch(data not shown) produced fluorescent intensities that were 18%, 39% and71% lower, respectively, than that achieved with the same samplesirradiated in the absence of electricity. Treatment with low levels ofelectricity (such as 1V) further diminished the stability of thessDNA:PNA hybrids containing bp mismatches. As the degree of sequencecomplementarity between the probe and the target decreased, the level offluorescent intensity diminished dramatically in the presence ofelectricity, providing a highly reliable and accurate second assay todifferentiate between perfectly matched sequences and those containing 1bp, 2 bp or 3 bp mutations.

Example 6

The hybridization assay in Example 5 was performed after denaturation ofthe dsDNA target sequences and measured ssDNA:PNA hybrid formation at atemperature above the melting point (T_(m)) of the dsDNA targets.Example 6 will demonstrate the reliability of the fluorescent intensityassay in the absence and presence of applied electricity todifferentiate between perfect matches and base pair mismatches withoutthe requirement for prior denaturation.

The hybridization reaction mixture (80 μl) contained the following: 4pmoles of target dsDNA, 4 pmoles of antiparallel PNA Probe No. 1,0.5×TBE and 250 nM of the DNA intercalator YOYO-1. Samples were placedinto a quartz cuvette, irradiated with an argon ion laser beam having awavelength of 488 nm for 80 msec and monitored for fluorescent emissionat 23° C. Concurrent temperature measurements were achieved by asoftware-controlled temperature probe placed directly into each sample.The maximum fluorescent intensity occurred at a wavelength of 536 nm,indicative of intercalation of YOYO-1 in the PNA:DNA hybrids. As asecond assay, following the initial laser irradiation of each sample,the same samples were subjected to 20V DC electrification for 4 seconds.Immediately after 3 seconds of electrification the samples wereirradiated a second time with the argon ion laser for 80 msec andmonitored for fluorescent emission at 23° C. Fluorescent intensitieswere plotted as a function of wavelength for each sample analyzed.

Enhanced by the intercalator YOYO-1, dsDNA:PNA triplexes were formed at23° C. The highest fluorescent intensity was achieved when the wild-type50-mer dsDNA target sequence (SEQ ID NO:1) was hybridized with the15-mer antiparallel PNA Probe No. 1 (FIG. 6). By comparison, thefluorescent intensities for a 1 bp mismatched dsDNA:PNA triplex (SEQ IDNO:2+Probe No. 1) and a consecutive 2 bp mismatched dsDNA:PNA triplex(SEQ ID NO:3+Probe No. 1) were 60% and 91% lower, respectively, thanthat observed with the perfectly matched dsDNA:PNA triplex at 23° C.(FIG. 6). When no DNA or PNA was present in the reaction mixturecontaining YOYO-1, only background levels of fluorescence were observed.

The difference in fluorescent intensities obtained by the perfectlycomplementary triplexes and those containing 1 bp or 2 bp mismatcheswere significantly greater than that achieved between perfectly matchedduplexes and incompletely complementary duplexes (compare FIGS. 5 and6). Clearly the fluorescent intensity assay of triplex formationpossessed enhanced discriminatory ability to detect base pairmismatches.

Moreover, even further discrimination between wild-type and mutatedsequences was possible with the secondary application of electricity. A20V treatment for 3 seconds to the perfectly matched dsDNA:PNA triplexesproduced a fluorescent intensity spectrum virtually identical to thatachieved by the same sample not subjected to electricity (FIG. 6).However, application of 20V for 3 seconds to the incompletelycomplementary triplexes containing a 1 bp mismatch and a 2 bp mismatchproduced fluorescent intensities that were 23% and 71% lower,respectively, than that obtained with the same samples irradiated in theabsence of electricity (FIG. 6). The 20V treatment of electricity didnot affect the stability of the perfectly complementary triplexes, butweakened the stability of the dsDNA:PNA triplexes containing base pairmismatches at a level dependent on the degree of sequencecomplementarity between the probe and the target. Therefore, theapplication of electricity to the fluorescent intensity assay providedan even more highly reliable assay to distinguish between wild-typesequences and those containing 1 bp or 2 bp mutations, without priordenaturation of sequences.

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made without departingfrom the spirit and scope thereof.

1. A method for assaying sequence-specific hybridization, said methodcomprising: providing a target comprising at least one target biopolymersequence; providing a probe comprising at least one probe biopolymersequence; adding said probe and said target to a binding medium toprovide a test sample; applying a first stimulus to said test sample toprovide a first stimulated test sample; detecting a first signal fromsaid first stimulated test sample, wherein said first signal iscorrelated with a binding affinity between said probe and said target;applying a second stimulus to said first stimulated test sample toprovide a second stimulated test sample; detecting a second signal fromsaid second stimulated test sample, wherein said second signal iscorrelated with said binding affinity between said probe and saidtarget; and comparing said first signal and said second signal toaccomplish said assaying; wherein: (a) at least one label is provided insaid test sample, (b) said first stimulus, said second stimulus, saidfirst signal and said second signal are photonic or electronic, (c) atleast one of said first stimulus and said second stimulus is photonic,(d) when said first stimulus and said second stimulus are photonic, anintermediate electronic stimulus is applied to said test sample aftersaid first stimulus and before said second stimulus, (e) said probebiopolymer sequence and said target biopolymer sequence containnucleobases and said probe hybridizes specifically with said target toform a homologous duplex, a homologous triplex, a homologous quadruplex,a Watson-Crick triplex or a Watson-Crick quadruplex, and (f) said methodis conducted without separating probe-target complexes from free probesand targets.
 2. The method of claim 1, wherein said first stimulus isphotonic and said second stimulus is electronic.
 3. The method of claim1, wherein said first stimulus is photonic and said second stimulus isphotonic.
 4. The method of claim 1, wherein said first stimulus iselectronic and said second stimulus is photonic.
 5. The method of claim1, wherein application of said second stimulus is at least partiallycoextensive with application of said first stimulus.
 6. The method ofclaim 1, wherein said first signal is photonic and said second signal iselectronic.
 7. The method of claim 1, wherein said first signal isphotonic and said second signal is photonic.
 8. The method of claim 1,wherein said first signal is electronic and said second signal isphotonic.
 9. The method of claim 1, wherein at least one of said firststimulus and said second stimulus is a laser beam.
 10. The method ofclaim 1, wherein said first stimulus, said second stimulus or saidintermediate electronic stimulus is electric voltage.
 11. The method ofclaim 1, wherein said at least one label transfers energy to at leastone other label to generate at least one of said first signal and saidsecond signal.
 12. The method of claim 1, wherein said at least onelabel is chemiluminescent or electrochemiluminescent.
 13. The method ofclaim 1, wherein said at least one label is an electron spin label. 14.The method of claim 1, wherein said probe hybridizes specifically withsaid target to form a homologous duplex substantially free of Hoogsteenbonding.
 15. The method of claim 1, wherein said probe hybridizesspecifically with said target to form a homologous or Watson-Cricktriplex substantially free of Hoogsteen bonding.
 16. The method of claim1, wherein said probe hybridizes specifically with said target to form ahomologous or Watson-Crick quadruplex substantially free of Hoogsteenbonding and free of G-G quartets.
 17. The method of claim 1, whereinsaid probe is a nucleic acid analog containing at least one of anuncharged backbone, a partially charged backbone, a cationic moiety, acrosslinking agent, a crosslinking sidechain and a nucleobase analog.18. The method of claim 1, wherein at least one of said probe biopolymersequence and said target biopolymer sequence contains an amino acidsequence.
 19. The method of claim 1, further comprising: applying atleast one additional stimulus to said second stimulated test sample toprovide an additionally stimulated test sample; detecting at least oneadditional signal from said additionally stimulated test sample, whereinsaid at least one additional signal is correlated with said bindingaffinity between said probe and said target; and comparing said firstsignal, said second signal and said at least one additional signal toaccomplish said assaying.
 20. The method of claim 19, wherein said firststimulus, said second stimulus and said at least one additional stimulusare different from each other.
 21. The method of claim 19, wherein atleast one of said first stimulus, said second stimulus and said at leastone additional stimulus is applied non-continuously.
 22. The method ofclaim 1, wherein at least one of said first stimulus and said secondstimulus is applied non-continuously.
 23. The method of claim 1, whereinat least one of said probe and said target is bonded to a substrate,surface, partition, membrane or electrode.
 24. The method of claim 1,wherein said at least one label is not covalently bound to said probe orsaid target.
 25. The method of claim 1, wherein said at least one labelis an intercalating agent.
 26. The method of claim 25, wherein said atleast one label is not covalently bound to said probe or said target.27. The method of claim 26, wherein said first stimulus is directlyapplied to said test sample and said second stimulus is directly appliedto said first stimulated test sample.
 28. The method of claim 1, whereinsaid first stimulus, said second stimulus or said intermediateelectronic stimulus is electric voltage applied to said test sample for15 seconds or less.
 29. The method of claim 1, wherein said firststimulus, said second stimulus or said intermediate electronic stimulusis electric voltage effective to destabilize imperfectly matchedhybridization partners and ineffective to destabilize perfectly matchedhybridization partners.
 30. A method for assaying sequence-specifichybridization, said method comprising: providing a target; providing aprobe, wherein at least one of said probe and said target comprises atleast one biopolymer sequence; adding said probe and said target to abinding medium to provide a test sample; applying a first stimulusdirectly to said test sample to provide a first stimulated test sample;detecting a first signal from said first stimulated test sample, whereinsaid first signal is correlated with a binding affinity between saidprobe and said target; applying a second stimulus directly to said firststimulated test sample to provide a second stimulated test sample;detecting a second signal from said second stimulated test sample,wherein said second signal is correlated with said binding affinitybetween said probe and said target; and comparing said first signal andsaid second signal to accomplish said assaying; wherein: (a) at leastone label is an intercalating agent provided in said test sample and isnot covalently bound to said probe or to said target, (b) said firststimulus, said second stimulus, said first signal and said second signalare photonic or electronic, (c) at least one of said first stimulus andsaid second stimulus is photonic, (d) when said first stimulus and saidsecond stimulus are photonic, an intermediate electronic stimulus isapplied to said test sample after said first stimulus and before saidsecond stimulus and (e) said method is conducted without separatingprobe-target complexes from free probes and targets.
 31. The method ofclaim 30, wherein at least one of said probe and said target is aprotein, a peptide or a lipid membrane.
 32. The method of claim 30,wherein one of said probe or said target is not a biopolymer.
 33. Themethod of claim 30, wherein said first stimulus, said second stimulus orsaid intermediate electronic stimulus is electric voltage applied tosaid test sample for 15 seconds or less.
 34. The method of claim 30,wherein said first stimulus, said second stimulus or said intermediateelectronic stimulus is electric voltage effective to destabilizeimperfectly matched hybridization partners and ineffective todestabilize perfectly matched hybridization partners.